The functional objectives of this project are to investigate genetic molecular structure of pathogens, to define the role of gene products in pathogenic mechanisms and to perform studies directed toward development of vaccines using molecular biology. Our primary emphasis focuses on cloning and expression of genes relevant to the toxic components of Bordetella pertussis. Pertussis toxin is the protective component of the whooping cough vaccine; however, its biological activities may cause the harmful side effects associated with current vaccines. This protein toxin is composed of five dissimilar subunits; the S1 subunit contains an ADP-ribosyltransferase activity responsible for most if not all of the biological activities of the toxin. Past accomplishments of the laboratory include: cloning and sequencing the pertussis toxin operon (patent application filed and licensed for vaccine development); direct expression of toxin subunits in Escherichia coli; and characterization of structure and function relationship between pertussis toxin, pathogeneses, and immunoprotection. Using the results of these studies as a basis for further research, we have identified a region in the S1 subunit that is required for enzymatic activity and that contributes to the formation of a neutralizing antigenic determinant. The gene encoding the S1 subunit was modified in this critical region by sitespecific mutagenesis. One of the mutants in which a single amino acid substitution was engineered into the S1 subunit had no enzymatic activity while retaining the protective epitope. The genetic changes responsible for the lost of enzymatic activity are now being engineered into the chromosome of B. pertussis to develop a new strain of the bacteria which will produce a genetically detoxified pertussis toxin. This modified toxin has important potential for use as a component of an acellular vaccine against whooping cough and is the subject of two invention reports and patent applications to be filed this year.